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Immunofluorescence Microscopy and mRNA Analysis of Human Embryonic Stem Cells (hESCs) Including Primary Cilia Associated Signaling Pathways

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This chapter describes the procedures for immunofluorescence microscopy (IFM) and quantitative PCR (qPCR) analyses of human embryonic stem cells (hESCs) grown specifically under feeder-free conditions. A detailed protocol is provided outlining the steps from initially growing the cells, passaging onto 16-well glass chambers, and continuing with the general IFM and qPCR anlysis. The techniques are illustrated with results on cellular localization of transcriptional factors and components of the Hedgehog, Wnt, PDGF, and TGFβ signaling pathways to primary cilia in stem cell maintenance and differentiation. Furthermore, a sample qPCR experiment is experimentally shown illustrating that these techniques can be important tools in answering basic questions about hESC biology.

OriginalsprogEngelsk
TidsskriftMethods in molecular biology
Vol/bind1307
Sider (fra-til)123-140
ISSN1064-3745
DOI
StatusUdgivet - 2016

ID: 44996656